Anti-salmonella antibodies and uses thereof

ABSTRACT

The present disclosure provides anti-Salmonella antibodies or antibody fragments such as camelid single domain antibodies (VHHs), along with associated nucleic acids, host cells and phages. Methods of reducing the presence of Salmonella in an animal or an animal environment, methods and formulations for treating Salmonella infection, and methods of detecting Salmonella are also described.

FIELD OF THE INVENTION

The field of the present invention relates generally to antibodies,fragments thereof, derivatives thereof, and to uses and applications ofsuch antibodies. The antibodies and fragments described may bespecifically directed against Salmonella.

BACKGROUND ART

Salmonellosis is one of the most commonly reported zoonotic diseases inhumans. In the United States alone, it causes an estimated 1.3 millionhuman food-borne illnesses and more than 500 deaths each year (Messenset al., 2013). Salmonella serotypes enteritidis and typhimurium arefrequently detected in human infections (Ravel et al., 2010).Salmonellas are widely distributed in nature, and they are commonlycarried by wild or farm-animal vectors. Poultry is known to be a majorglobal reservoir of Salmonellas. Salmonella live in poultry gut astransient members of the intestinal microbial population without causingdisease. Colonization of Salmonella does not usually affect poultry bodyweight gain or performance; thus, asymptomatic infection can increasethe likelihood of zoonotic transmission to humans through the food chain(Hugas et al., 2014; Mazengia et al., 2014). Chicks can become infectedvertically (from adults via the egg to the chick) or horizontally (fromthe environment, pests, or feed) (Cox et al., 2014; Rodriguez et al.,2006).

Salmonella enterica is one of the two main Salmonella species thatcauses gastroenteritis in humans. S. enterica is subdivided into 6subspecies and almost all human infections are caused by subspecies I(enterica). More than 2600 serovars of S. enterica have been identified(Popoff and Le Minor, 1997); however, only a few of these serovars areresponsible for most Salmonella infections in human and domestic animals(Porwollik et al., 2004).

The large and growing market for broiler chickens and eggs, and theemergence of antibiotic resistant strains of Salmonella have led topublic health concerns, change in government regulation policies inEurope and North America and further demands to enact laws to controlSalmonella levels in poultry (Hugas, et al., 2014).

Vaccination strategies in broiler chickens have shown sub-optimalresults to-date mostly due to the short life span of the birds.Currently, two types of Salmonella vaccines are commercially available;an attenuated live vaccine and an inactivated vaccine. These vaccinesare often administered to both breeder and layer flocks, but theireffectiveness depends on the targeted serovar, host species, and whetherreduction rather than eradication is the objective (Doyle and Erickson,2006). These vaccines do not eliminate initial colonization of themucosal surfaces, particularly in the young bird (Dougan et al., 1988).Effective control depends upon a number of factors, including improvedon-farm biosecurity, use of best practices in husbandry and use ofvaccination and competitive exclusion products and feed additives.Preventive hygienic measures typically involve establishing effectivefarm-site biosecurity and poultry house sanitation protocols. Other moretargeted strategies are being developed. For instance, a combination ofSalmonella-specific lytic phages has been recently approved in Europefor applications in food packaging. Others have proposed and testedinclusion of bacteriocins and/or tailspike phage protein(Chakchouk-Mtibaa et al., 2014; Waseh et al., 2010) in the poultry feedfor controlling Salmonella but to date none of these products has beencommercialized.

It would be advantageous to provide antibodies or fragments thereof thatassist in the reduction, prevention and/or treatment of Salmonellainfection.

SUMMARY OF THE INVENTION

This disclosure refers to the development of camelid single domainantibodies (VHHs) that bind to Salmonella. VHHs are the smallest antigenbinding fragments that can be readily expressed in bacteria or yeast inlarge quantities and at a significantly lower cost compared toconventional antibodies.

Accordingly, disclosed herein is an isolated antibody or antibodyfragment comprising an amino acid sequence of any one of SEQ IDNOS:1-18, or a variant thereof. In one embodiment, the isolated antibodyor antibody fragment binds directly to the exterior of Salmonella,optionally to Salmonella flagella.

In a preferred embodiment of the present disclosure, theSalmonella-binding antibody or antibody fragment comprises a CDR1comprising an amino acid sequence of GRX₁FSX₂KP; a CDR2 comprising anamino acid sequence of ASX₃TGVST; and a CDR3 comprising an amino acidsequence of AGTX₄RTLWGSKWRDX₅X₆EYEY; wherein X₁ is T or S; X₂ is V or K;X₃ is F or Y; X₄ is T or L; X₅ is V or R; and X₆ is L or R.

In another preferred embodiment, the Salmonella-binding antibody orantibody fragment comprises a CDR1 comprising an amino acid sequence ofGLDFSSYA; a CDR2 comprising an amino acid sequence of ISRFGGRL; and aCDR3 comprising an amino acid sequence of AADRRSGLGTSKEYDY.

In another preferred embodiment, the Salmonella-binding antibody orantibody fragment comprises a CDR1 comprising an amino acid sequence ofGIIFSINA; a CDR2 comprising an amino acid sequence of ISAYDHT; and aCDR3 comprising an amino acid sequence of NVDEIRKF.

In another preferred embodiment, the Salmonella-binding antibody orantibody fragment comprises a CDR1 comprising an amino acid sequence ofGRSFSLYG; a CDR2 comprising an amino acid sequence of ISGSGLATS; and aCDR3 comprising an amino acid sequence of AQRWTSGTIARATGEYGY.

In another preferred embodiment, the Salmonella-binding antibody orantibody fragment comprises a CDR1 comprising an amino acid sequence ofGSIFSGDA; a CDR2 comprising an amino acid sequence of IGKEGDT; and aCDR3 comprising an amino acid sequence of ATFEERPQPSYVY.

In a preferred embodiment of the present disclosure, the isolatedantibody or antibody fragment disclosed herein is modified for toleranceto one or more gut enzymes selected from the group consisting of pepsin,trypsin and chymotrypsin. In a further preferred embodiment, theantibody or antibody fragment disclosed herein comprises a detectablelabel.

The present disclosure further provides a nucleic acid molecule encodingthe isolated antibody or antibody fragment disclosed herein, a host cellcomprising the nucleic acid molecule, and a bacteriophage comprising thenucleic acid or the polypeptide.

Another preferred aspect of the present disclosure is a method ofreducing the presence of Salmonella in an animal or an animalenvironment comprising administering to the animal the isolated antibodyor antibody fragment disclosed herein. In one preferred embodiment, themethod further comprises administering an antibiotic, bacteriocin, orother plant- or animal-derived compound effective against Salmonella tothe animal. In another preferred embodiment, the method furthercomprises administering a competing microbe to the animal together withan antibody or antibody fragment disclosed herein, optionallyco-expressed or co-contained in a probiotic system. The antibody orantibody fragment may be administered orally; the animal may be achicken, optionally a laying hen or broiler chicken; and the animalenvironment may be a poultry farm.

Also disclosed is a method of reducing or preventing introduction ofSalmonella into an animal environment comprising administering to aninductee animal the antibody or antibody fragment disclosed herein,prior to introducing the inductee animal into the animal environment.

Also disclosed is a method of treating a Salmonella infected subjectcomprising administering to the subject the isolated antibody orantibody fragment disclosed herein. In a preferred embodiment, themethod of treating an infected subject further comprises administeringto the subject antibiotic effective against Salmonella. The subject maybe a livestock animal selected from the group consisting of a chicken,cow, or sheep, or the subject may be a human.

Also disclosed is a formulation for use in treating Salmonella infectioncomprising the isolated antibody or antibody fragment disclosed hereinand a pharmaceutically acceptable excipient.

Further disclosed is a use of the isolated antibody or antibody fragmentdisclosed herein for treating Salmonella infection in a subject in needthereof.

Further disclosed is a method of detecting Salmonella in a samplecomprising contacting the sample with the isolated antibody or antibodyfragment disclosed herein, and detecting the presence of bound antibodyor antibody fragment. In one preferred embodiment, the sample comprisesa bodily fluid or fecal material. In another preferred embodiment, thesample comprises a food product or a surface swab from a food product.

Another aspect of the present disclosure is a kit for conducting thedetection method, comprising the isolated antibody or antibody fragmentdisclosed herein and instructions for use in detecting Salmonella.

Another aspect provides use of the antibody or antibody fragmentdisclosed herein for preparation of a medicament for treatment ofSalmonella infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects and advantages will become apparent from the followingdescription taken together with the accompanying drawings in which:

FIGS. 1A-1F show exemplary amino acid sequences of the anti-Salmonellaantibodies and antibody fragments of the present disclosure and nucleicacid sequences encoding said antibodies and antibody fragments;

FIG. 2 is a dendrogram showing amino acid sequence similarities betweenthe anti-Salmonella antibodies and antibody fragments;

FIG. 3 shows purified anti-Salmonella VHH domains on SDS-PAGE gelstained with Coomassie Brilliant Blue™ G250;

FIG. 4 is a bar graph showing results of a binding assay for 18different anti-Salmonella VHH domains to flagellin of Salmonella orwhole cells of Salmonella enterica strains: S. Heidelberg 918, S.Heidelberg 4643, S. Hadar 5643, or S. Hadar 5659 (binding measured atA450);

FIG. 5 is a bar graph showing the results of a motility assay for S.enterica strain SG4904 in the presence of 4 different anti-SalmonellaVHH domains (y-axis shows the diameter of the outgrowth of bacterialcolonies);

FIG. 6 is a bar graph showing the results of a cell proliferation assayof S. enterica strain SG4904 in the presence of 4 differentanti-Salmonella VHH domains (bacterial growth rate determined bymeasuring OD₆₀₀);

FIG. 7 is a bar graph showing the activity of three VHHs against S.enterica serovar Hadar 5643 in a HeLa cell Salmonella internalizationassay;

FIG. 8 is a bar graph showing the activity of different concentrationsof three VHHs against S. enterica serovar Hadar 5643 in a culturedchicken ileum or jejunum Salmonella internalization assay, as measuredby Salmonella genome copy number (based on amplification of the singlecopy housekeeping gene ttr); and

FIG. 9 is a scatter plot showing the effects of administering VHH 0A07to non-SPF broiler chicks challenged with S. enterica serovar Hadar 5643(CFU counts for ileum and jejunum sections are shown).

DETAILED DESCRIPTION

The present disclosure is based on the creation, isolation, andcharacterization of antibodies and antibody fragments that preferablyhave the ability to bind to the exterior of Salmonella cells. Featuresand uses of said antibodies and antibody fragments will now be describedin greater detail. It will be appreciated that exemplary embodimentspresented herein are within the scope of the present invention and arenot intended as limiting. Reference is made to the Figures which relateto preferred embodiments of the present invention.

Definitions

The term “antibody” as used herein refers to a full lengthimmunoglobulin that has the ability to bind to an antigen. The term“antibody fragment” as used herein refers to a less than full lengthportion of an immunoglobulin molecule for example, a VHH domain whichretains the ability to bind to an antigen.

The term “VHH” or “VHH domain” as used herein refers to a single domainantibody derived from a heavy chain antibody raised in a camelid animal,such as a llama, alpaca, or camel. Other terms for VHHs sometimes usedin the art include but are not limited to: single domain antibodies(sdAbs), single variable domain antibodies, immunoglobulin singlevariable domains, heavy-chain variable domain antibodies, andNanobodies™.

The term “isolated” or “purified” as used herein in association with apolypeptide, antibody, antibody fragment or a nucleic acid means apolypeptide, antibody, antibody fragment or nucleic acid that issubstantially or essentially free of naturally associated molecules—forexample, an isolated antibody that is substantially or essentially freeof antibodies having different specificities.

The term “multimeric” or “multivalent” as used herein refers to havingmultiple antigen-binding locations on a polypeptide, typically frommultiple copies of an antibody or antibody fragment, or from a pluralityof similar but different such antibodies or antibody fragments.

The term “nucleic acid” as used herein refers to double stranded orsingle stranded DNA, RNA molecules or DNA/RNA hybrids. These moleculesmay be nicked or intact as found in living cells. The double stranded orsingle stranded nucleic acid molecules may be linear or circular. Theduplexes may be blunt ended or have single stranded tails, for example,with sticky ends created by restriction endonucleases.

The term “variant” as used herein refers to an amino acid or nucleotidesequence having at least 80% identity or sequence homology with asubject amino acid or nucleotide sequence.

Antibodies and Antibody Fragments

According to one aspect of the present disclosure, provided herein is anisolated antibody or antibody fragment comprising an amino acid sequenceof any one of SEQ ID NOS:1-18, or a variant thereof. In a preferredembodiment, said antibody or antibody fragment binds to Salmonella. In afurther preferred embodiment, said antibody or antibody fragment bindsto the flagella of Salmonella. The aforementioned antibodies or antibodyfragments are preferably derived from a collection of antibodies raisedin alpacas that were immunized with heat inactivated Salmonella cells,as described in these Examples below. Each of SEQ ID NOS:1-18corresponds to the amino acid sequence of an isolated VHH domain fromsaid collection and said sequences are shown in FIGS. 1A, 1B, 1C, 1D, 1Eand 1F.

VHH domains are comprised of framework regions interspersed with threecomplementarity determining regions (CDRs): CDR1, CDR2, and CDR3. CDRsequences are known to be essential for the specificity of bindingbetween antibodies (or antibody fragments) and antigens. Accordingly, ina preferred embodiment of the present disclosure, the antibody orantibody fragment disclosed herein comprises a CDR1 comprising an aminoacid sequence of GLDFSSYA (SEQ ID NO:40); a CDR2 comprising an aminoacid sequence of ISRFGGRL (SEQ ID NO:41); and a CDR3 comprising an aminoacid sequence of AADRRSGLGTSKEYDY (SEQ ID NO:42). In another preferredembodiment, CDR1 comprises an amino acid sequence of GIIFSINA (SEQ IDNO:43); CDR2 comprises an amino acid sequence of ISAYDHT (SEQ ID NO:44);and CDR3 comprises an amino acid sequence of NVDEIRKF (SEQ ID NO:45). Inanother preferred embodiment, CDR1 comprises an amino acid sequence ofGRSFSLYG (SEQ ID NO:46); CDR2 comprises an amino acid sequence ofISGSGLATS (SEQ ID NO:47); and CDR3 comprises an amino acid sequence ofAQRWTSGTIARATGEYGY (SEQ ID NO:48). In a further preferred embodiment,CDR1 comprises an amino acid sequence of GSIFSGDA (SEQ ID NO:49); CDR2comprises an amino acid sequence of IGKEGDT (SEQ ID NO:50); and CDR3comprises an amino acid sequence of ATFEERPQPSYVY (SEQ ID NO:51).

Consensus sequences can be defined based on some of the above-specifiedCDR sequences, as described in the Examples below and illustrated inTable 2. In a preferred embodiment of the present disclosure, anisolated antibody or antibody fragment is herein provided that bindsSalmonella and comprises a CDR1 comprising an amino acid sequence ofGRX₁FSX₂KP (SEQ ID NO:37); a CDR2 comprising an amino acid sequence ofASX₃TGVST (SEQ ID NO:38); and a CDR3 comprising an amino acid sequenceof AGTX₄RTLWGSKWRDX₅X₆EYEY (SEQ ID NO:39); wherein X₁ is T or S; X₂ is Vor K; X₃ is F or Y; X₄ is T or L; X₅ is V or R; and X₆ is L or R.Optionally, CDR1 comprises an amino acid sequence of GRTFSVKP (SEQ IDNO:52); CDR2 comprises an amino acid sequence of ASFTGVST (SEQ IDNO:53); and CDR3 comprises an amino acid sequence ofAGTTRTLWGSKWRDVLEYEY (SEQ ID NO:54). Optionally, CDR1 comprises an aminoacid sequence of GRSFSVKP (SEQ ID NO:55); CDR2 comprises an amino acidsequence of ASFTGVST (SEQ ID NO:53); and CDR3 comprises an amino acidsequence of AGTLRTLWGSKWRDRREYEY (SEQ ID NO:56). Optionally, CDR1comprises an amino acid sequence of GRTFSKKP (SEQ ID NO:57); CDR2comprises an amino acid sequence of ASYTGVST (SEQ ID NO:58); and CDR3comprises an amino acid sequence of AGTTRTLWGSKWRDVLEYEY (SEQ ID NO:54).

Any of the antibody fragments described herein may be utilized in anisolated form, or may form a portion of a longer molecule, such aswithin an antibody, for example a recombinant antibody, a chimericantibody, a small molecule conjugated antibody, a human antibody, or ahumanized antibody.

Furthermore, an antibody fragment may include, but is not limited to Fv,single-chain Fv (scFv; a molecule consisting of V_(L) and V_(H)connected with a peptide linker), Fab, Fab′, and F(ab′)2, or singledomain antibody (sdAb). SdAbs may be of camelid origin, and thus may bebased on camelid framework regions; alternatively, the CDRs may begrafted onto the framework regions of other antibody domains, forexample but not limited to VNAR, human V_(H) or human V_(L) frameworkregions.

The present invention includes modifications of the antibodies orantibody fragments disclosed herein, and may include amino acidvariations, including conservative substitutions, additions ordeletions, provided at least 80%, preferably at least 90%, identity orsequence homology is observed and provided such a modification resultsin a functional variant. In a preferred embodiment, the percent identityis set at 90% or greater, and thus it is to be understood that anidentity of each VHH domain can, for example, be individually determinedas 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of specifiedsequences.

It will be appreciated that the present antibodies or antibody fragmentscan also be preferably produced in multimeric forms. For example, adimer or pentamer can be formed. Antibody fragments, such as VHHs, thatare used to form a multimer may be the same or different from eachother. Pentavalent multimeric VHH domains, or pentabodies, may possesshigher affinity binding to an antigen as compared with monovalent VHHdomains. The five VHH domains need not be identical to one another, andas such may comprise VHH domains of different sequences.

The antibodies or antibody fragments described herein may preferably bemodified for tolerance or resistance to one or more gut enzymes. Typicalgut enzymes which may have a destructive effect on a polypeptide includepepsin, trypsin and chymotrypsin. Thus, resistance to these enzymes isadvantageous, as the peptide would have more exposure time to bind withambient Salmonella within the intestinal tract. Single domain antibodiesare, in general, significantly more resistant to proteases thanconventional antibodies. Furthermore, VHHs are known to be amenable topolypeptide engineering for optimization of biophysical featuresincluding heat and protease resistance (Hussack et al., 2014). Scaffoldengineering of portions of the polypeptide both inside and outside ofthe CDR regions are known in the art and can confer increased targetaffinity, protease resistance, as well as thermal and low pH resistance.For example, to favour the entropy of binding, the extended flexibleCDR3 loop may be constrained with an interloop disulfide bond thatconnects CDR1 and CDR3, or CDR2 position 55 and CDR3 or FR2 at position50 and CDR3 (Muyldermans, 2013; Conrath et al, 2003). The cysteine ofCDR3 that participates in either disulfide bond formation mentionedabove can occur or be placed at any position of the extended CDR3 loop(Conrath et al, 2003). The stability of a VHH can be increased byintroducing cysteine at position 54 and 78 to form an additionaldisulfide bond. This disulfide bond is known to make VHHs highlyresistant to degradation by pepsin or chymotrypsin (Hagihara et al,2007; Saerens et al, 2008; Hussack et al, 2014).

The antibodies or antibody fragments described herein may preferably belabeled with an acceptable label and optionally a linker as needed. Thelabel may be rendered detectable or may in itself be detectable, so thatthe presence of binding to Salmonella can be observed. The antibody orantibody fragment may be linked to a radioisotope, a paramagnetic labelsuch as gadolinium or iron oxide, a fluorophore, Near Infra-Red (NIR)fluorochrome or dye, an echogenic microbubble, an affinity label (forexample biotin, avidin, etc), enzymes, or any other suitable agent thatmay be detected by diagnostic imaging methods.

The antibodies or antibody fragments described herein may be produced inany of the ways known in the art. For example, antibodies or antibodyfragments may be expressed in a cell containing an encoding expressionvector. Such expression systems are well known in the art and manyvariations may be used. Examples of cell-based expression systems may beSaccharomyces cerevisiae, Bacillus subtilis, Bacillus brevis, Bacillusmegaterium, Lactobacillus species, Escherichia coli, Pichia pastoris,Aspergillus niger, and mammalian-derived cell lines such as CHO, HEK, orHeLa cells. The expressed antibody or antibody fragment can be isolatedfrom a solution of lysed cells or the polypeptide may be secreted intomedia and isolated directly therefrom. The antibody or antibody fragmentmay also be artificially synthesized. Examples of artificial proteinsynthesis include solid-phase peptide synthesis, liquid-phase peptidesynthesis, and cell-free protein synthesis, also known as in vitroprotein synthesis.

Nucleic Acids, Cells, and Bacteriophages

Nucleic acid molecules encoding the amino acid sequences described aboveare encompassed herein. In one embodiment, the nucleic acid moleculecomprises a nucleic acid sequence of any one of SEQ ID NOS:19-36. Thesesequences are illustrated in FIGS. 1A, 1B, 1C, 1D, 1E and 1F. Given thedegeneracy of the genetic code, a number of variant nucleic acidsequences would have the effect of encoding the amino acid, as would bereadily understood by one skilled in the art. The nucleic acid moleculesof the present invention may be double stranded or single stranded DNA,RNA molecules or DNA/RNA hybrids. These molecules may be nicked orintact as found in living cells. The double stranded or single strandednucleic acid molecules may be linear or circular. The duplexes may beblunt ended or have single stranded tails, for example, with sticky endscreated by restriction endonucleases.

A host cell comprising the nucleic acid molecule encoding any one of theantibody or antibody fragment described herein would also be readilyrecognized by the skilled artisan. The host cell could be a bacterium,such as a desired strain of E. coli, a yeast cell, such as a desiredstrain of Pichia pastoris, a mammalian-derived cell line such as CHO,HEK, or HeLa cells, or any other host cell suitable for carrying thenucleic acid molecule of the present invention.

A bacteriophage comprising the antibody or antibody fragment describedherein, and/or comprising the nucleotide molecule encoding the antibodyor antibody fragment is also encompassed in the present invention.

Methods, Uses, Formulations, and Kits

The antibodies or antibody fragments described herein can specificallybind to Salmonella and it will be appreciated that said antibodies orantibody fragments may be useful for a number of purposes, includingreducing Salmonella, inhibiting Salmonella and/or detecting Salmonellain a subject.

Accordingly, provided herein are methods of reducing the presence ofSalmonella in an animal or an animal environment comprisingadministering to the animal an antibody or antibody fragment disclosedherein.

Within an individual animal, reducing the presence of Salmonella maycomprise reducing contamination on the surface of the animal, or withinthe gastrointestinal tract of an animal. Should an animal besystemically infected, the method described herein could be used forreducing the presence of Salmonella.

The environment of an animal can preferably relate to the animal'simmediate surroundings, such as the walls or floors of a cage orfacility, the feeding or watering apparatuses within an animal compound,the bedding materials found in an animal compound, or simply the fecalmaterial present external to the animal within the animal's confines.

Administering to an animal the antibody or antibody fragment describedherein can preferably be for the purpose of reducing or inhibiting thepresence of Salmonella within the animal to which the antibody orantibody fragment is administered, or an offspring of such an animal, orwithin the flock, cage or barn in which the animal lives. ReducingSalmonella within the animal's gastrointestinal tract is one way toreduce contamination within the animal's environment, leading to a saferfood supply chain with lower incidence of contamination.

Co-administration of another substance that is effective againstSalmonella is also a possible strategy for reducing Salmonella in ananimal environment. For example, administering to the animal anantibiotic either at the same time as a co-formulation or at an adjacenttime to the delivery of the antibody or antibody fragment can have anadditive effect or may have a synergistic effect. The result of whichmay be a reduced likelihood of Salmonella contamination, but alsoreduced usage of antibiotic (i.e., fractional usage of antibiotic withsynergistic efficacy). A bacteriocin effective against Salmonella canalso be provided to the animal with the antibody or antibody fragmentfor an additive or synergistic effect. In addition to, or as analternative to bacteriocin, any other plant- or animal-derived compound,such as a small molecule, peptide, or protein, that has an effectagainst Salmonella may be used together with the antibody or antibodyfragment described herein. A competitive microbe may also be provided tothe animal concurrently with the antibody or antibody fragment in orderto possibly achieve an additive or a synergistic effect. The competitivemicrobe may be used together with the antibody or antibody fragmentdescribed herein as part of a probiotic system. Within such a probioticsystem, the antibody or antibody fragment may be co-administered withthe competitive microbe, or may be delivered sequentially. Expression ofthe antibody or antibody fragment within a probiotic system of thepolypeptide described herein may also be undertaken.

The antibody or antibody fragment described herein may be administeredorally to a subject. Oral delivery permits the polypeptide to bedelivered within the water or food supply to an animal, and is lessnoticeable or stressful to an animal than an injection. Gavage is alsoan acceptable oral route when highly accurate delivery of an oral dosingregime is desirable. Other routes of administration can also beconsidered, such as inhalation, intranasal, gel-based or by spray, inovo, topically or by injection such as intravenous, subcutaneous,intramuscular, intraorbital, intraocular, intradermal, gel-based, sprayor rectal delivery route. The antibody or antibody fragment may beadministered directly or within a phage or host microorganism.

As described above, modifications to the antibody or antibody fragmentdescribed herein may be made to increase efficacy of oral delivery. Forexample, scaffold engineering can be performed on portions of theantibody or antibody fragment within or outside of the CDR regions toconfer protease resistance, as well as thermal and low pH resistance.However, and in addition, the form of antibody or antibody fragmentdelivery may also be altered with pharmaceutically acceptable coatingsor excipients that provide a protective effect against gut enzymes,thermal or low pH effects. In this way, the sequence of the antibody orantibody fragment itself need not be modified, but rather theformulation prepared for oral delivery may itself be more optimal forthe species of subject to which the antibody or antibody fragment is tobe delivered. The antibody or antibody fragment may also be conjugatedto small molecules such as cyclic peptides, macromolecules orpolyethylene glycol to improve delivery or stability.

The dosage form may be of any type acceptable for antibody or antibodyfragment delivery to animals. Coated forms and slow release forms couldbe used if desirable. Liquid, powder, crystal, gel, semi-solid, ortablet forms can be used.

The animal to which the antibody or antibody fragment may be deliveredmay preferably be a bird, such as a broiler chicken or laying hen. Othertypes of livestock animals, such as swine, cows, sheep, etc. may alsobenefit from the peptide if Salmonella is present in the animal's gut orsurrounding environment. A preferred animal environment may be a barn orfarm, such as a poultry farm.

In order to avoid contamination of an animal environment that issubstantially free of Salmonella, a method is provided that aims toprevent introduction of a new contaminated animal or “inductee” animalinto the environment, such as a barn. In such a method, the antibody orantibody fragment is administered to an inductee prior to introducingthe inductee animal into the animal environment, such as a barn or farm.In this way, the animal could be cleared for the likelihood ofcontamination prior to taking up residence with the other animals whomay have already received treatment.

The antibody or antibody fragment may also be administered to plants orplant-based materials by spraying or by other methods to reduce a levelof contaminating Salmonella. Examples of such plants or plant-basedmaterials include but are not limited to salads and spices.

Also disclosed is a method of treating a Salmonella infected subjectcomprising administering to the subject an antibody or antibody fragmentdisclosed herein. In a preferred embodiment, the method furthercomprises administering to the subject antibiotic effective againstSalmonella. The subject may be a livestock animal selected from thegroup consisting of a chicken, cow, or sheep, or the subject may be ahuman.

Also disclosed is a formulation for use in treating Salmonella infectioncomprising an antibody or antibody fragment disclosed herein and anexcipient.

Also disclosed is a use of an antibody or antibody fragment disclosedherein for treating Salmonella infection in a subject in need thereof.

Also disclosed is a method of detecting Salmonella in a samplecomprising contacting the sample with an antibody or antibody fragmentdisclosed herein, and detecting the presence of bound antibody orantibody fragment. In one preferred embodiment, the sample comprises abodily fluid or fecal material. In another preferred embodiment, thesample comprises a food product or a surface swab from a food product.

For detection purposes, samples from a subject may comprise a bodilyfluid or fecal material. The subject may be a human or a non-humananimal. Samples of microbiota can be collected from the gastrointestinal(GI) tract or gut of a subject. Methods of sample collection are knownto those skilled in the art. For example, microbiota samples may beobtained from stools, intestinal mucosal biopsies, gut lavage orcombinations thereof. Collection can be performed during endoscopy byflushing a physiological solution, such as sterile saline solution orsterile water, onto the mucosa to remove the strongly adherent mucuslayer overlying mucosal epithelial cells and the microbial communityembedded within the mucus layer. Aspirates are then collected directlythrough an endoscope at a specific location in the gut and the samplesare placed on ice.

Collection of gut microbiota can also be performed on stools. Collectionof bacteria from stools is known in the art. In the case of fecalmicrobiota collection and analysis, fresh stools may be collected,immediately processed, and the processed materials can be stored atabout −80° C.

For detection purposes, the subject may be a chicken, optionally abroiler chicken. Samples from such a subject may comprise intestinalfluid, carcass, feathers, skin, breast/leg meat rinses, as well asdroppings from poultry or a bodily fluid, or rectal effluent. Samplesmay be taken from the environment such as the floor covering of barns,boots, wash/chill tanks or any other equipment used at a poultryprocessing facility as well as animal feed and water.

Once a sample has been collected, the presence of bound antibody orantibody fragment in said sample can be detected by carrying out any oneof a number of binding assays or bound substrate detection proceduresknown in the art. Antibody or antibody fragment binding can be measureddirectly or indirectly by using a tagged version of the antibody orantibody fragment, examples of which are described above. The step ofdetecting may be accomplished by any suitable method known in the art,for example, but not limited to: optical imaging, immunohistochemistryor molecular diagnostic imaging, ELISA, or other suitable method.

Also disclosed is a kit for conducting the detection method, comprisingan antibody or antibody fragment disclosed herein and instructions foruse in detecting Salmonella.

Exemplary embodiments of the present disclosure will now be described.These embodiments involve preparation and use of camelid single-domainantibodies (VHHs) specific for Salmonella, and are not intended aslimiting.

EXAMPLES Example 1

Immunization of Alpaca with Different Strains of Salmonella

To isolate VHH domains that target Salmonella, three alpacas wereimmunized with different strains of Salmonella enterica.

Three male alpacas (Vicugna pacos) were immunized subcutaneously withSalmonella enterica serovars. Five injections were performed in total.Each animal was injected with a mixture of 4 Salmonella enetericastrains (1×10⁹ cfu from each) that were heat inactivated (30 min at 65°C.) and mixed with adjuvant (aluminum hydroxide, Alhydrogel™ 2%). Theinjection groups consisted of:

-   -   1—S. Typhimurium (SGSV1412, and SGSC4904), S. Entritidis        (SGSC4901, SGSC3820);    -   2—S. Newport (SGSC4910), S. Javiana (SGSC4917), S. Senftenberg        (SGSC2516), S. Heidelberg (SGSC4966); and    -   3—S. Hadar (SGSC4906), S. Kentucky (SGSC4914), S. Infantis        (SGSC4905), S. SaintPaul (SGSC4920).

To prepare antigens for injection, Salmonella were cultured on LB agarplates. Approximately 1×10⁹ cells were treated with heat at 65° C. for30 minutes in order to completely kill the bacteria. For each injection,a total of 4×10⁹ killed cells were used with equal mixture of 4different strains in a total volume of 0.4 ml. The contents were mixedwith an equal volume of Alhydrogel™ (Sigma™). Injections were done atdays 1, 15, 22, 29, and 36. Alpaca serum was analyzed for specificbinding to a commercially available purified flagellin from S.Typhimurium or the whole bacteria cells immobilized on the plates.Briefly, microtiter plates (Maxisorp™ plates) (Nalge NuncInternational™, Rochester, N.Y.) were coated overnight at 4° C. with 5μg/ml of flagellin antigen in PBS. Wells were rinsed and blocked with200 μl of 5 mg/ml Bovine Serum Albumin or 1% casein. Different dilutionsof serum were added and incubated at room temperature for 1.5 h. Wellswere washed with PBST (0.05% v/v Tween-20TH), and incubated with goatanti-llama IgG (H+L) (1:1,000 in PBS) (Bethyl Laboratories™, Montgomery,Tex.) followed by Rabbit-anti-goat-HRP (1:5,000 in PBS) (BethylLaboratories™, Montgomery, Tex.). Signal was detected by adding 100μl/well TMB peroxidase substrate (Kirkegaard and Perry Laboratories™,Gaithersburg, Md., USA). Reactions were stopped by adding 100 1Mphosphoric acid and A450 was measured using a Bio-Rad™ ELISA platereader.

Example 2

Phage Display Library Constructions

A hyper-immunized alpaca VHH library was constructed based on RNAisolated from the lymphocytes of animals immunized as in Example 1.

A phage display library was constructed using a standard protocol(Arbabi Ghahroudi et al., 2009). Lymphocytes were collected from theblood using Lymphoprep™ Tubes (Axis-Shield™, Oslo, Norway). Total RNAwas isolated from approximately 1×10⁷ lymphocytes collected on day 36post-immunization using RNAzol™ kit (Bioshop™′ Burlington, Ontario,Canada). First-strand cDNA was synthesized with oligo(dT) primers fromthe SuperScript III First Strand™ cDNA synthesis kit (Invitrogen™,Burlington, Ontario, Canada) using 6 μg total RNA as template accordingto manufacturer's recommendations. Variable and part of the constantdomains DNA were amplified using oligonucleotides MJ1-3 (sense) and twoCH2 domain antisense primers, CH2 and CH2b3 (for primer sequences seeArbabi Ghahroudi et al., 2009; Baral et al. 2013) and heavy chainfragments (550-650 bp in length) were purified using the E.Z.N.A.™ CyclePure PCR purification Kit (Omega Bio-tek™ Norcross, Ga., USA). Thevariable regions of heavy chain antibodies (IgG2 and IgG3) werere-amplified in a second PCR reaction using MJ7 and LP6-MJ8 primers (forprimer sequences, see Baral et al. 2013). The amplified PCR productswere purified with the Cycle Pure Kit™ (Omega Bio-tek™), digested withSfil (Thermoscientific™, Toronto, Ontario, Canada), and re-purifiedusing the same kit. Twelve micrograms of digested VHH fragments wereligated with 40 μg (1:3 molar ratio, respectively) Sfi-digested pADL-23cphagemid vector (Antibody Design Labs™, San Diego, Calif., USA) using T4DNA ligase system and its protocol (Promega™, Madison, Wis., USA),transformed into commercial electrocompetent TG1 E. coli cells (Lucigen™Corporation, Middleton, Wis., USA), as described previously (ArbabiGhahroudi et al., 2009), and a library size of 7.8×10⁸ transformants wasobtained. The VHH fragments from 50 colonies were PCR-amplified andsequenced to analyze the complexity of the library; all clones hadinserts of expected sizes and were different from each other at theirCDR regions as determined by sequencing of their encoding VHH fragments.The library was grown for 3-4 hours at 37° C., 250 rpm in2×YT/Carb-Glucose (1% w/v) medium. The bacterial cells were pelleted,resuspended in the same medium and stored as glycerol stock at −80° C.as described previously (Arbabi Ghahroudi et al., 2009).

Example 3

Screening Phage Display Library to Select for VHHs Binding to Salmonella

The library screening (panning) was done through a sequential strategyusing either whole, heat inactivated Salmonella bacteria or purifiedSalmonella flagellin protein (main component of flagella encoded by fliCgene) as a target. For the panning against whole Salmonella, thebacterial cells from 4 different strains were equally mixed and adjustedto an OD of 1 using PBS solution. The bacterial mixes were inactivatedat 65° C. for 30 minutes and coated on a 96 well Maxisorb™ plate. To panagainst flagellin protein (Flagellin from Salmonella typhimurium,purchased from Sigma™, cat #: SRP8029), flagellin solution (5 ug/ml,dissolved in PBS solution) was coated on a 96 well Maxisorp™ plate. Foreach panning, BSA-PBS solution (0.5% of BSA dissolved in PBS solution)was also coated on the same 96-well Maxisorp™ plate as a pre-screeningcontrol. The coated plates were then incubated at 4° C. overnight. Nextday, the coated wells were rinsed with PBS once and blocked with BSA-PBSsolution for 2 h at 37° C. The Salmonella phage library was dilutedusing BSA-PBS solution so that approximately 2×10¹² phage particles wereadded first to the BSA-PBS wells and kept at 37° C. for 1 hour and thenthe supernatant was transferred to the Salmonella whole cell orflagellin wells. The phage particles were incubated in the Salmonellawhole cell or flagellin wells for 2 hours at 37° C. and then washed 5times with PBST containing 0.1% v/v Tween-20. The bound phages wereeluted with 0.1 M triethylamine, neutralized with 1M Tris-HCL, PH 7.4and incubated with exponentially growing TG1 cells in 10 ml of 2YTmedium. After 30 min incubation at 37° C., the cells were superinfectedwith 10¹¹ M13KO7 helper phage (New England Biolabs™) for an additional30 min. Two antibiotics, ampicillin (100 μg/L) and Kanamycin (25 μg/L),were added to the TG1 culture medium. Incubation continued at 37 QC for16 hours, followed by selection of the phage infected TG1 cells. In thethird day of panning, the amplified phage particles in culturesupernatant were precipitated with polyethylene glycol (PEG) asdescribed previously (Arbabi-Ghahroudi et al., 2009). Briefly, 10 ml ofphage infected TG1 culture was centrifuged at 4000 rpm at 4° C. for 30minutes, the supernatant was filtered with a 0.22 μm filter and mixedwith 1/5 volume of PEG/NaCl (20% PEG, 2.5 M NaCl) on ice for 1 hour. Thephages were pelleted by centrifugation at 4000 rpm at 4° C. for 30minutes. Finally, the enriched phage pool was suspended in 200 μl of PBSand ready for the next round of panning. Panning was continued for threemore rounds following the same conditions except that washing wasincreased 7, 10 and 12 times with PBST for the second, third and fourthrounds of panning, respectively. After four rounds of panning, 96randomly picked colonies were grown and subjected to phage ELISAscreening.

Example 4

Expression and Purification of Monomeric VHH

VHH against flagellin or whole Salmonella cells identified in Example 3were PCR amplified from the pADL23 phagemid vector with BbsI1-VHHforward primer (5′-TATGAAGACACCAGGCCCAGGTAAAGCTGGAGGAGTCT-3′) (SEQ IDNO:59) and BamHI-VHH reverse primer(5′-TTGTTCGGATCCTGAGGAGACGGTGACCTG-3′) (SEQ ID NO:60). The PCR fragmentswere digested with the BbsI and BamHI restriction enzymes and ligatedinto the similarly digested pSJF2 expression vector (Arbabi-Ghahroudi etal., 2009). Upon ligation, all plasmids were transformed intoelectrocompetent E. coli (TG1 strain) and selected on LB agar platescontaining carbenicillin. Colonies were screened by colony PCR forinserts and the DNA was sequenced. The sequences were aligned andcategorized into 18 different groupings or classes; each represented byone or more clones and each representing the same amino acid sequence(Table 1). A single clone was randomly selected from each class. Nucleicacid and amino acid sequences of the 18 selected VHHs are shown inFIG. 1. CDR1, CDR2, and CDR3 are underlined within each polypeptidesequence. As shown in Table 2, the 18 VHH sequences can be placed in 5distinct groups based on sequence similarities in the CDR regions.

The VHHs all have the canonical amino acid residues found in Camelidfamily VHHs at positions 42 (F/Y), 49 (E/Q/A), 50 (R), and 52 (F/V/G/L)(Muyldermans, et al. 1994) according to the IMGT numbering scheme(Lefranc, et al. 2003). In addition, the CDR3 domains in these VHHs aregenerally larger than CDR3 domains in human VH proteins. For example,the CDR3 domain of Group 1 VHHs is 20 residues in size (Table 2).

Analysis using the full polypeptide sequences of the 18 VHHs revealsthat these VHHs can be classified into 2 larger groupings (FIG. 2). Thefirst grouping includes 1E05, 1E03, 1H07, 4D01, 0D12, 1A07, 1608, 0A07,0H12 and 0A08; the second grouping includes 4E08, 4F12, 1E08, 3B04,2A09, 1G06, 0A09 and 4C10.

VHH antibodies were expressed using the standard periplasmic expressionmethod (Arbabi-Ghahroudi et al., 2009). VHH antibody 1E03 was expressedin P. pastoris because no expression was achieved using the E. coliexpression method. After induction of protein expression, cell cultureswere harvested at 6,000 rpm×30 min (4° C.), the supernatant decanted,and the periplasmic contents extracted from the cell pellet. Briefly,the pellet of monomeric VHH was resuspended in 20 ml of ice cold TES(0.2 M Tris-HCl pH 8.0, 20% (w/v) sucrose, 0.5 mM EDTA) and incubated onice for 30 min. Next, 30 ml of ice-cold 1/8 TES (diluted in dH₂O) wasadded, incubated an additional 30 min on ice, and the slurry centrifugedat 9,000 rpm for 30 min (4° C.). The resulting supernatant containingVHH was dialysed overnight against PBS and purified using Profinity™IMAC resin from BioRad™, as per manufacturer's instructions, withphosphate-based elution buffer containing 500 mM imidazole.

Purified protein fractions were pooled and dialyzed against PBS. Elutedfractions were analyzed by SDS-PAGE and Western blotting before beingdialysed into PBS. FIG. 3 shows an example of VHH polypeptides on anSDS-PAGE gel stained with Coomassie™ Brilliant Blue G250. The arrowpoints to the position of the polypeptides. Size of the polypeptides isabout 24 kD. Expected size is around 15 kD. The discrepancy is caused bythree tags (c-myc, AviTag™ and His₆) linked to the VHHs.

VHH concentrations were determined by absorbance measurements at 280 nmusing theoretical MW and extinction coefficients calculated with theExPASy ProtParam™ Tool (expasy.org/tools/protparam.html) according toPace et al., 1995. The yield of the purified monomeric VHHs ranged from1 to 20 mg/L bacterial culture.

Example 5

VHH Binding Assays

Microtitre plates (Maxisorp™ plates) (Nalge Nunc International™,Rochester, N.Y.) were coated overnight at 4° C. with 2.5 μg/mL flagellin(Sigma-Aldrich™ SRP8029) and 14 different heat inactivated (65° C., 30min) Salmonella enterica serovar whole cells. Binding of flagellin and 4of the S. enterica strains is described in Example 1. Wells were rinsedwith PBS pH 7.4 and blocked with 200 ul of BSA-PBS solution for 1 hr.Twenty different VHH-containing phages were prepared as described inExample 1 and used to test their binding ability to flagellin or 14different Salmonella strains. Phages in 2YT medium supernatant (1004)were added to blocked coated microtitre plates and incubated at roomtemperature for 1 hour. Wells were washed with PBST solution andincubated with HRP conjugated anti-M13 monoclonal antibody (1:5000 inPBST) (Sigma-Aldrich™ GE27-9421-01) at room temperature for half anhour. Wells were washed again and signal was detected using 50 ul/wellTMB peroxidase substrate (Kirkegaard and Perry Laboratories™,Gaithersburg, Md., USA). Reactions were stopped by adding 50 ul/well of1M hydrochloric acid and A450 was measured using a Cytation™ 5 (Biotek™)multimode reader. As shown in FIG. 4, a number of the VHHs weredetermined to bind to one or more of Salmonella flagellin and/or theSalmonella serovar whole cells. For example, robust binding was observedfor VHH 1E05 against all inactivated S. Hadar and S. Heidelberg strainstested. This assay provides only a qualitative or semi-quantitativeassessment of the target binding of the VHHs. Furthermore, bindingcannot be discounted for VHHs that were not show to bind according tothis assay, as said assay is limited by the presentation of antigen oncoated plates and by the variation in growth rates of phages displayingparticular VHHs.

Example 6

Salmonella Motility Assays

Motility assays was performed as described previously (Kalmokoff et al.,2006). VHHs at a final concentration of 2 μg/μl were plated withSalmonella strain 4904 (1×10⁷) on Muller-Hinton™ media with 0.35% agarand incubated at 37° C. under microaerophilic conditions (5% O₂, 10% CO₂and 85% N₂) for 18 hours. Bacterial motility was determined by measuringthe diameter of the circle produced by the growing bacteria on theplate. Results are shown in FIG. 5. It was found that VHH 1E08significantly inhibited bacterial motility.

Example 7

Cell Proliferation Assays

Around 1×10⁷ Salmonella (strain name: 4904) cells were incubated in 100μl Muller-Hinton™ liquid medium with 0.2 μg/μl VHHs in a 96 well plateand incubated at 37° C. under microaerophilic conditions (5% O₂, 10% CO₂and 85% N₂) for 18 hours. Bacterial growth rate was determined bymeasuring optical density at 600 nm wavelength (FIG. 6). Similar to themotility assay result, VHH 1E08 was found to significantly inhibit thegrowth of Salmonella cells.

Example 8

Salmonella Internalization Assays in HeLa Cells

Aim:

to identify anti-Salmonella VHH domains that interfere with Salmonellacolonization of epithelial cells. To colonize the gut, Salmonella mustattach to the surface and enter the host epithelial cells, where itundergoes intracellular replication. Without wishing to be limited bytheory, VHHs that interfere with this process are expected to preventbacterial attachment and/or allow attachment but block the invasion ofthe host cell.

Summary of the Assay:

HeLa epithelial cells are challenged with GFP expressing Salmonella inthe absence or presence of different VHHs. Salmonella is allowed toattach and enter the cells and subsequently unbound bacteria areremoved. Attached but non-internalized Salmonella are eliminated bygentamicin, an antibiotic that does not penetrate epithelial cells.Intracellular growth of Salmonella is then tracked by using afluorescence plate reader, which quantifies the increase of GFPfluorescence over time.

Method:

HeLa cells were grown in 24-well plates containing 500 μl/well ofDMEM+10% FBS and incubated at 37° C. in the presence of 5% CO₂ for 24 h(80-100% confluence). Salmonella enterica serovar Hadar 5643 transformedwith GFP was grown on the same day (from an overnight culture) to an ODof approximately 0.5. The culture was centrifuged (5 min, 5000 rpm) andresuspended in DMEM without FBS, and the bacteria were pre-incubatedwith the VHHs at different concentration for 30 min at 37° C. withgentle mixing. Prior to infection, cells were washed 3× with PBS.Salmonella and VHHs were added to the HeLa cells (MOI: 100 in 500ul/well of DMEM without FBS). The plates were incubated for 1 h at 37°C. in 5% CO₂. After 1 h of infection (BEFORE GENTA samples—FIG. 7) cellswere washed 3× with PBS to remove non-adherent bacteria. To permeabilizeand lyse HeLa cells, 1 ml of 1% saponin was added. The plates wereincubated for 15 min at 37° C. in 5% CO₂. Bacteria were resuspended bypipetting up and down vigorously (approx. 10 times per well). Serialdilutions were plated on LB agar plates. The plates were incubatedovernight at 37° C. to quantify viable intracellular bacteria. DMEM with10% FBS+gentamicin (100 μg/ml, final conc.) was added to the rest of theplate. After 1 h of infection (AFTER GENTA samples-FIG. 7) cells werewashed 3× with PBS to remove non-adherent bacteria. To permeabilize andlyse HeLa cells, 1 ml of 1% saponin was added. The plates were incubatedfor 15 min at 37° C. in 5% CO₂. Bacteria were resuspended by pipettingup and down vigorously (approx 10 times per well). Serial dilutions wereplated on LB agar plates. The plates were incubated overnight at 37° C.to quantify viable intracellular bacteria. DMEM with 10% FBS+gentamicin(10 ug/ml, final conc.) was added to the rest of the plates and theywere incubated for 24 h at 37° C. in 5% CO₂. All assays were performedin duplicate.

Results:

Based on both colony counts and fluorescence quantification of GFPexpression, VHH 0A07 showed the highest inhibition of intracellular S.enterica strain Hadar 5643 growth (FIG. 7).

Example 9

Salmonella Internalization Assays in Chicken Ileum and Jejunum Cultures

Aim:

to identify anti-Salmonella VHHs that interfere with Salmonellacolonization of adult chicken intestine. To colonize the chick gut,Salmonella must attach to the surface and enter the host epithelialcells, where it undergoes intracellular replication. Without wishing tobe limited by theory, VHHs that interfere with this process are expectedto prevent bacterial attachment and/or allow attachment but block theinvasion of the host cell.

Method:

Intestinal jejuni and ilea were obtained from ten (2×5) 30-day oldnon-SPF chickens. The jejuni and ilea were washed and cut into 0.5×0.8cm pieces. VHHs 0A07, 0A08, 1E03, 1H07 and 0H12 were pre-bound to S.enterica serovar Hadar 5643 for 30 min. The mixture was applied onto theintestinal sections and the bacteria were allowed to infect for 3 hours.Treatment with gentamicin removed extracellular bacteria. Genomic DNAwas extracted from the infected sections and an established robust 5′nuclease (TaqMan™) real-time PCR assay was used to detect Salmonella aspreviously described (Malorny et al., 2004).

Results:

7.5-log reduction in genome copy number was achieved with the 0A07, 1E03VHH treatment at 50 μg/mL for the S. enterica serovar Hadar 5643infection of ileum sections (FIG. 8). 6-log reduction in genome copynumber was also achieved with the 1E03 VHH treatment on infection ofjejunal sections. 4-log reduction in genome copy number was observedwith the 1H07 VHH on infection of jejunum sections. No MIC wasdetermined because no complete inhibition was achieved up to 50 μg/mL.The results represent biological duplicates.

Example 10

Treatment of Broiler Chickens with VHHs

The objective of the study presented in this Example was to test theefficacy of VHH 0A07 in a Salmonella enterica challenge model byadministration via oral gavage.

Method:

Animal testing and data collection was carried out by Colorado QualityResearch, Inc. (Wellington, Colo.). Commercial broiler chickens (Cobb500 breed) were supplied by Simmons Foods™ (Siloam Springs, Ark.).Chicks were received at about 1 day old and put on a non-medicatedindustry average diet. The birds were housed in concrete floor penswithin an environmentally controlled facility. All birds were placed inclean pens containing clean pine shavings as bedding. Lighting was viaincandescent lights and a commercial lighting program was used. Waterand feed was provided ad libitum throughout the study. The testfacility, pens and birds were observed at least twice daily for generalflock condition, lighting, water, feed, and ventilation. Upon receiptand prior to placement, all birds were tagged in the back of the neckwith uniquely numbered individual identification tags. Clinicalobservations of all birds were made once daily. These observationsincluded body weight and feed intake measurements.

For the study, 60 one-day old chicks were randomly assigned to one oftwo treatment groups, Group A or Group B each group consisting of 30birds. Group A was the control group; birds within this group receiveduntransformed, inactivated E. coli cells. In contrast, Group B birdsreceived inactivated E. coli expressing VHH 0A07.

Upon arrival, the chicks were tagged, randomized, weighed, and placedinto 6 pens (2 blocks of 3 pens; 10 birds per pen). The followingmorning, the birds were challenged by oral gavage. Specifically, birdswere gavaged with 0.5 ml of S. enterica at a concentration of 1.0×10⁸CFU/bird. Treatment was thereafter administered three times via oralgavage: at 1 h, 24 h, and 48 h post challenge gavage. Birds in Group Awere gavaged with inactivated E. coli; Group B with inactivated E. coliexpressing VHH 0A07 (1.5 mg per dose per bird in 1000 dH₂O). At 70 h,birds were weighed and euthanized.

Tissue samples were immediately collected from the euthanized birds formicrobiological assays. Jejunum and ileum materials were composited intoone sample per bird. Collected samples were transported on icepacks toMicrobial Research Inc. (Fort Collins, Colo.) for microbiologicalassays.

Result:

Administration of VHH 0A07 significantly decreased the levels ofSalmonella enterica serovar Hadar 5643 in chick jejunum and ileum by 0.7log₁₀ (p=0.029) (FIG. 9). The geometric mean and median of Salmonellaload in control Group A (not expressing VHH 0A07) was 2.2×10⁴ and3.1×10⁴, respectively. The geometric mean and median of Salmonella loadin Group B (expressing VHH 0A07) was 5.2×10³ and 1.2×10⁴, respectively.

Tables

TABLE 1 Anti-Salmonella VHHs The following are the numbered clonesidentified within each class of VHHs. Class Colony 1 4E08, 4E12, 4F10,4G04, 4G10, 4H05 2 4A03, 4A04, 4A07, 4A09, 4A11, 4B01, 4B02, 4H01, 1E083 0A09 4 1E10, 0C07, 4C09, 4D02, 4D03, 1C11, 0A11, 2C07, 0A08, 0F11,0G08, 0G12 5 0G11, 1B08 6 1A08, 1C10, 0A07, 0B08, 0B10, 0B11, 0C11,0E09, 0E11, 0F12, 0H10 7 1E05 8 2A09 9 0E07, 1B07, 1C08, 1D07, 1D08,1F10, 1G08, 0A10, 0A12, 0C10, 0D07, 0D09, 0D10, 0E08, 0E12, 0F07, 0G09,0H11, 0H12 10 3B04, 3B07, 4A05, 4A06, 4E02, 4E06, 4E10, 4E11, 4F02,4F03, 4F06, 4F09, 4F11, 4G05, 4G06, 4G08, 4G09, 4G11, 4H02, 4H06, 4H08,4H11, 4H12, 0B07 11 1H07, 0F10, 2A02, 1E11, 3A01, 3A02, 3A03, 3A04,3A05, 3A06, 3A07, 3A08, 3A09, 3A10, 3A11, 3B01, 3B02, 3B03, 3B05, 3B06,3B08, 3B09, 3B10, 3B11, 4A01, 4A12, 4B04, 4B05, 4B06, 4B07,, 4B08, 4B09,4B10, 4B11, 4B12, 4C01, 4C02, 4C03, 4C04, 4C05, 4C06, 4C07, 4C08, 4C11,4C12, 4D04, 4D05, 4D06, 4D07, 4D08, 4D09, 4D10, 4D12, 4E01, 4E03, 4E04,4E05, 4E07, 4E09, 4F01, 4F04, 4F05, 4F07, 4F08, 4G02, 4G03, 4G07, 4G12,4H03, 4H04, 4H07, 4H09, 4H10, 1C09, 1F07, 1F08, 2H05, 0B12, 0D11, 0E10,0F08, 0G07, 0H07, 0H09, 12 1B06, 1E03 13 1G06, 2A07, 2B02, 0C08 14 1G03,2H10, 0C09, 0D08, 0F09, 0G10, 2B07, 1A07 15 4C10, 4G01 16 4D01, 4D11 174F12 18 0D12

TABLE 2 Consensus amino acid sequences of CDR1, CDR2 and CDR3 of the five groups of anti-Salmonella VHHs CDR1 Group 1 GRX₁FSX₂KP0A08; 1B08; 0A07;  1E05; 0H12; 1H07; 1E03; 0F09; 4D01;  0D12 Group 2GLDFSSYA 4F10; 1E08; 3B04;  4F12 Group 3 GIIFSINA 2A09; 1G06 Group 4GRSFSLYG 0A09 Group 5 GSIFSGDA 4C10 X₁ = T or S; X₂ = V or K  CDR2Group 1 ASX₃TGVST 0A08; 1B08; 0A07;  1E05; 0H12; 1H07;1E03; 0F09; 4D01;  0D12 Group 2 ISRFGGRL 4F10; 1E08; 3B04;  4F12 Group 3ISAYDHT 2A09; 1G06 Group 4 ISGSGLATS 0A09 Group 5 IGKEGDT 4C10 X₃ =F or Y CDR3 Group 1 AGTX₄RTLWGSKWRDX₅X₆EYEY 0A08; 1B08; 0A07; 1E05; 0H12; 1H07; 1E03; 0F09; 4D01; 0D12 Group 2 AADRRSGLGTSKEYDY4F10; 1E08; 3B04;  4F12 Group 3 NVDEIRKF 2A09; 1G06 Group 4AQRWTSGTIARATGEYGY 0A09 Group 5 ATFEERPQPSYVY 4C10 X₄ = T or L; X₅ =V or R; X₆ = L or R

The scope of the claims should not be limited by the preferredembodiments set forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole.

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What is claimed is:
 1. An isolated antibody or antibody fragment thatbinds to Salmonella comprising a Complementary Determining Region(CDR)1, a CDR2 and a CDR3, wherein the CDR1, CDR2 and CDR3 comprise thefollowing: (i) the CDR1 comprising an amino acid sequence of GLDFSSYA(SEQ ID NO:40), (ii) the CDR2 comprising an amino acid sequence ofISRFGGRL (SEQ ID NO:41), and (iii) the CDR3 comprising an amino acidsequence of AADRRSGLGTSKEYDY (SEQ ID NO:42).